Consistency of Product Quality for SB5, an Adalimumab Biosimilar. Peaks are assigned by three symbols, a legend can be found on the bottom left. BPCs of adalimumab at resolution settings 17,500, 35,000 and 70,000. Epub 2022 Jan 20. Support high throughput analysis with 15 minute separation and enable detailed characterization on charge variant peaks of multiple monoclonal antibodies without redundant method development or fraction collection. BPC of adalimumab, acquired at a resolution setting of 35,000. No records were found matching your criteria, Faster, Easier, and Better Biopharmaceutical Charge Variant Profiling, Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Biopharmaceutical Characterization Information, Spectroscopy, Elemental and Isotope Analysis, Protein Aggregate and Subvisible Particles Analysis Information, pH gradients for charge variant separations, Get Greater Structural Insight into Biotherapeutic Proteins: Charge Variants and Aggregates, The advantage of pH gradient buffers is demonstrated by the ion exchange separation of charge variants of denosumab, Separation of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography, High Salt Gradient Analysis of Post-Translational Modifications- Deamidation Monitoring, A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis, A Novel pH Gradient Separation Platform for Monoclonal Antibody (MAb) Charge Variant Analysis, High-Resolution Charge Variant Analysis for Top-Selling Monoclonal Antibody Therapeutics Using a Linear pH Gradient Separation Platform, High-Resolution Separation of Intact Monoclonal Antibody Isoforms, High-Throughput, High-Resolution Monoclonal Antibody Analysis with Small Particle Size HPLC Columns, Utilizing the Native Fluorescence of Monoclonal Antibodies for the Sensitive Detection of Charge Variants, High-Resolution Separation of a Fusion Protein, Monoclonal Antibody Analysis on a Reversed-Phase C4 Polymer Monolith Column, Separation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC Column, Separatoion of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography, Using the NISTmAb reference standard to demonstrate a simple approach to charge variant analysis, Evaluation and Application of Salf- and pH-Based Ion-Exchange Chromatography Gradients for Analysis of Therapeutic mAbs, Simple Charge Variant Profile Comparison of an Innovator mAb and a Biosimilar Candidate, Automated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of Variants, Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic interaction Chromatography (HIC) Columns, Advantages of High-Resolution Separation Media for Monoclonal Antibody Analysis, Advantages of Small Particle High-Resolution Separation Media for Monoclonal Antibody Analysis, Development of a Cation-Exchange pH Gradient Separation Platform, Intact Mass Analysis of Monoclonal Antibody (MAb) Charge Variants Separated Using Linear pH Gradient, MAbPac SCX 3 and 5 m Particle Phases for Monoclonal Antibody (MAb) Variant Analysis, Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns, Small Particle Media for High Throughput, High Resolution Monoclonal Antibody Analysis, Novel Ways to Introduce the Traditional Salt Based Chromatography Techniques of Size Exclusion and Ion Exchange Chromatography of Biopharmaceutical Proteins Into High Resolution Mass Spectrometry, New chromatographic workflows for charge variant profiling, intact mAb analysis and DAR determination, Development of Ultra-fast pH-Gradient Ion Exchange Chromatography for the Separation of Monoclonal Antibody Charge Variants, Characterizing Therapeutic Monoclonal Antibodies, Bringing Biosimilar Therapeutics To Market Faster A CRO Case Study. BPC of adalimumab, acquired at a resolution setting of 35,000. High-resolution separation and Identification of proteoforms with as little as 0.02 pI value difference. A.) sharing sensitive information, make sure youre on a federal The site is secure. Each fragment (Fragments 1, 2, and 3) has different characteristics. Instrument settings and gradients used for all mAbs are provided in the methods section. This technote is focused Intabio is being used under license. Rx Only. 2021 Oct 20;13(11):1747. doi: 10.3390/pharmaceutics13111747. One such critical attribute that requires monitoring during cell-line and process development for a given biologic drug candidate is its charge profile. This can be overcome through the use of commercially available gradient buffers which, in combination with the correct column, produces a linear gradient. 2022 Jan;10(1):e003171. FOIA 2018 Sep 15;1095:166-176. doi: 10.1016/j.jchromb.2018.07.037. Bookshelf Charge Variant Analysis. The https:// ensures that you are connecting to the Before Revealing charge heterogeneity of stressed trastuzumab at the subunit level. The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. FOIA Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies. This difference is observed as a difference in charge variants with a slight acidic shift of Fragment 2 (Samples C and D) (Fig 6). Bio IEX-HPLC columns are designed for high resolution, high recovery and efficient separations of biological compounds, such as oligonucleotides, proteins, and peptides. Fragment 1 has several charge variants between pH 6.5 and 7.8, and Fragment 2 in Sample B has several charge variants between pH 5.9 and 6.5. doi: 10.1186/bcr3069. In anion-exchange, positively charged surface ligands interact with negatively charged analytes; in cation-exchange, negatively charged surface ligands interact with positively charged analytes. After the IEF, Cy3 and Cy5 multiplex fluorescence images of samples separated on the Immobiline DryStrip gels were captured using ImageQuant 800 imager in fluorescence mode (Fig 3). Epub 2023 Jan 25. Reduce the overall analytical testing burden during biotherapeutic development with streamlined and informative workflows using Multiple Attribute Methodology. Ltd. or their respective owners in the WebThorough characterization of monoclonal antibodies (MAb) includes the identification and monitoring of acidic and basic isoforms. Milln-Martn S, Carillo S, Fssl F, Sutton J, Gazis P, Cook K, Scheffler K, Bones J. Eur J Pharm Biopharm. Gain advantages in data quality and time with an integrated system that offers high-resolution and high-throughput separation of intact mAbs and their charge variants. Join scientific experts from the National Institute of Bioprocess Research and Training NIBRT in Dublin, Ireland and Thermo Fisher Scientific to hear about the latest analytical advances for intact protein characterization. Spanov B, Olaleye O, Mesurado T, Govorukhina N, Jungbauer A, van de Merbel NC, Lingg N, Bischoff R. Anal Chem. Accelerate candidate selection by achieving separation, quantitation, and direct identification of biopharmaceutical charge variants and their proteoforms with icIEF-UV/MS via the Intabio ZT system. Unexpectedly, a C4 400 column (3.4 m diameter with 0.2 m porous shell) provided higher peak capacities compared to the same 1000 column (2.7 m diameter with 0.5 m porous shell). Unauthorized use of these marks is strictly prohibited. Pharmaceuticals (Basel). Capillary zone electrophoresis, with its unique mode of action, brings key Proteins have an inherent charge under any given set of conditions, and this can be altered by changes to the proteins primary structure, or by the addition of post-translational modifications (PTMs). Harness the ability to transform rich data into results. The characterization of charge heterogeneity and determination of isoelectric point (pI) adds a critical dimension to establishing identity, purity, post-translational modification and stability of therapeutic protein preparations. New columns and instrument technologies have shown the potential to decrease the run times even further: shorter small particle size columns combined with linear pH gradients can bring this analysis into the domain of a true UHPLC application. Collect the data for comprehensive charge variant ID in minutes not weeks. Differential regulation of human monocytes and NK cells by antibody-opsonized tumors. Challenge the status quo with identification of charge variant peaks in minutes not weeks on a single platform. The charge variant peak profiles changed with time and the peak area percentages for all variants for the Unstressed, Stressed-2W, and Stressed-4W samples are summarized in Table 1. Effect of Trastuzumab-HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab. Protein charge variants can be separated using physicochemical fractionationbased on charge characteristics of antibodies or other proteins. Gradient from 0100% buffer B in 10 minutes followed by 4 minutes, A.) IEF was carried out on 24 cm pH 310 (linear) Immobiline DryStrip gels and second dimension with a large format gel with low fluorescent glass plates (DIGE gels). Unable to load your collection due to an error, Unable to load your delegates due to an error. Top: the results of IEF and corresponding 2D patterns of Samples A, B, C, and D are shown in 2D view with pI calibration. sharing sensitive information, make sure youre on a federal This enables method optimization to be achieved easily and logically and delivers robustness and consistency between operators and laboratories. official website and that any information you provide is encrypted Charge variant separation of seven monoclonal antibodies using a gradient from 0100% buffer B in 10min. Cruz E, Sifniotis V, Sumer-Bayraktar Z, Reslan M, Wilkinson-White L, Cordwell S, Kayser V. Pharmaceutics. J Chromatogr B Analyt Technol Biomed Life Sci. Two sets of experiments with 7 cm pH 3-10 NL (Non-linear) and 24 cm pH 3-10 L (linear) Immobiline DryStrip gels was carried out. The site is secure. A Comparison of Infliximab Therapeutics in Regard to Charge Variant Profile and Antigen Binding Affinity. Interpreting their structural differences can take weeks and requires the use of multiple analytical methods and instruments, increasing the complexity of the charge heterogenity analysis assay, and creating a bottleneck in gaining critical information for processes and team members downstream in the development pipeline. The fragments were joined by disulfide bounds (Table 1), thereby forming a homo- or a heterodimer. Leblanc Y, Faid V, Lauber MA, Wang Q, Bihoreau N, Chevreux G. J Chromatogr B Analyt Technol Biomed Life Sci. Fig 4. C.) Relative abundance of the 3 most intense glycoforms of all 3 lysine variants identified. 2021 Jan;158:83-95. doi: 10.1016/j.ejpb.2020.11.006. The development of a universal method based on pH elution was explored using a number of mAb drug products. official website and that any information you provide is encrypted Decrease the chance of product stability or safety issues, drive process development projects to completion or enable the study of more conditions during cell line development with an integrated icIEF-UV/MS workflow. Webcharge variants. PMC MeSH detection. 8600 Rockville Pike -, Liu H, Gaza-Bulseco G, Faldu D, Chumsae C, Sun J. Table showing the mass differences between the main glycoform of the three acidic species and the major lysine variant (no C-terminal lysine residues and G0F/G0F glycoform). Data from the mass analysis confirmed the potential modifications in both acidic and basic variant. Disclaimer. We further explored and adapted the chromatographic buffer system to expand the application range. The ligand-independent HER2-HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival. Trastuzumab has a pI range of 8.7-8.9 and is composed of mixture of acidic and basic variants beside the main peak. Trastuzumab has a pI 2016;8:15481560. A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. The analysis ensures product consistency with no unwanted changes to the protein. Jeong SA, Choi JM, Park JM, Lee JY, Lee SJ, Lee SY, Lee SY, Park YA, Jeong HJ, Song YC, Kim SH, Chang SJ. The process of identifying individual charge variant components is time and labor intensive. Collect the data for comprehensive charge variant ID in minutes not weeks. and transmitted securely. rather than conventional ionic strength salt gradients (90 min.). C.) BPCs of samples incubated at elevated temperature for up to 11days. Bethesda, MD 20894, Web Policies C.) Magnification of charge state +26 with labels of the three most abundant isoforms. Discovery chief, David Zaslav, was clear from the day he took control of CNN in 2022 about what he wanted for the Losacco GL, Hicks MB, DaSilva JO, Wang H, Potapenko M, Tsay FR, Ahmad IAH, Mangion I, Guillarme D, Regalado EL. Careers. available in all countries. Charge ahead with charge variant analysis and proteoform identification in minutes on a single platform. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. It was found that mAbs with isoelectric points in the range of around 79 could be well separated using cation exchange columns operated in a pH-based elution mode. As covered in Scheme 1, this report focuses on a) the key methods for physical separation of mAb charge variants, b) structural analysis of the fractions with mass including associated logos, are the property of AB Sciex Pte. WebThe Intabio ZT system couples icIEF separation and UV detection with high resolution mass spectrometry on the ZenoTOF 7600 system. Epub 2018 Jul (1) Beyer, B., Walch, N., Jungbauer, A., How Similar is Biosimilar? Experience one integrated workflow with a single, accessible dataset containing the required information to make the right decisions, fast. The workflow is greatly simplified by the use of a platform method, resulting in reduction or elimination of method optimization and easier scale up for deployment in routine environments. Accessibility Confidently select lead candidates and ensure reproducible product quality. Pte. doi:10.1038/nrd3746. Disclaimer. 2023 DH Tech. The advantages of pH-based gradients include a global applicability of the method to any monoclonal antibodies (mAb), greatly simplifying the method development. 2021 Feb;413(5):1267-1277. doi: 10.1007/s00216-020-03089-3. Overall, these data has provided meaningful insights to the impact of antibody charge variants on in vitro potency and CD16 binding affinity of trastuzumab. Confidently detect and fully identify charge variants during cell-line and early product/process development phases with optimized workflows that speed up timelines without compromising quality. Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems Trappe, Florian Fssl 1 , Silvia Figure 7 shows the 2D analysis on the 24 cm pH 310 (linear) Immobiline DryStrip gels offering better resolution than 7 cm pH 310 (Non-linear) Immobiline DryStrip gels. The electric field is re-oriented to initiate the mobilization of the peaks toward the electrospray The .gov means its official. Use. Ostrowski MA, Mack S, Ninonuevo M, Yan J, ElNaggar M, Gentalen E, Michels DA. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. A.) Unauthorized use of these marks is strictly prohibited. Bookshelf Berkowitz SA, Engen JR, Mazzeo JR, Jones GB.. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Published by Elsevier B.V. Spanov B, Baartmans B, Olaleye O, Nicolardi S, Govorukhina N, Wuhrer M, van de Merbel NC, Bischoff R. Anal Bioanal Chem. The Intabio ZT system couples icIEF separation and UV detection with high resolution mass spectrometry on the ZenoTOF 7600 system. Federal government websites often end in .gov or .mil. It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. Not Sell My Data, Comprehensive characterization of charge variant-associated quality attributes, Charge variant analysis throughout development, High-resolution charge variant monitoring, Rapid screening assays throughout cell line development. J Pharm Biomed Anal. For example, NanoPro technology coupled with photochemical immobilization and chemiluminescence technology could be used for high throughput measurement of the charge heterogeneity of mAb products (Michels et al., In order to make decisions early in product development there is a need for confident detection of charge variants, but also to fully identify what those variants are in a timeline keeping with the fast-pace of biotherapeutic development. Epub 2011 Nov 30. Quickly and confidently accelerate candidate selection by achieving separation, UV quantitation, and direct identification of biopharmaceutical charge variants and their proteoforms. HPLC or UHPLC also have global applicability and familiarity in all biopharmaceutical laboratories. Rapid protein therapeutic charge variant characterization across a wider range of modalities. In Samples C and D, Fragment 1 is located in the lower molecular weight region. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation. Clipboard, Search History, and several other advanced features are temporarily unavailable. Epub 2018 Jul 29. Measuring charge variants to ensure a consistent production process for biologics. Covid 19, Privacy Published by Elsevier Ltd. All rights reserved. for peak identification. Captured image data was further analyzed by Melanie 9 2D image analysis software. Wagons are a small but important segment in Europe. Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation. After the electrophoresis, gel images were captured using Amersham ImageQuant 800 Western blot imaging system and Amersham Typhoon biomolecular imager. Fragments 2 and 3 differ as the O-glycosylation site present in Fragment 3 was removed by a charge neutral mutagenesis in Fragment 2 resulting in different post-translational modifications. An ultra-fast RP-LC method with a run time of one minute was developed using trifluoroacetic acid which was later replaced by formic acid as additive for better MS compatibility. WebCreative Biolabs now offers comprehensive mAb charge variant analysis services to isolate and identify different charged variant forms using different charged-based The .gov means its official. B.) These large heterogeneous molecules can be subject to a variety of enzymatic post-translational modifications during manufacture, such as glycosylation and lysine truncation. Webinar: HCP-MS Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms. PMC National Library of Medicine Every time. acidic and basic variants). Take back your time with fast, easy to use CZE solutions that are simple to transfer globally. official website and that any information you provide is encrypted An official website of the United States government. Achieve the full picture and accelerate decisions with comprehensive characterization of biopharmaceutical charge variants. Images were scanned using Amersham Typhoon laser scanner, and analyzed using Melanie 9 software. The .gov means its official. Bookshelf 2022 Jun 3;11(2):19. doi: 10.3390/biotech11020019. 2023 Feb 28;95(8):3951-3958. doi: 10.1021/acs.analchem.2c03275. Moreover, poor reproducibility of salt-based gradients meant that method replication and robustness was often poor and method development was time consuming. Vitro Diagnostic The main peak in blue corresponds to adalimumab without both C-terminal lysine residues, orange represents the variant containing one C-terminal lysine and green the variant carrying both C-terminal lysine residues. 2022 Mar;6(3):215-231. doi: 10.1038/s41570-021-00353-7. Simplified Methods Use the same method over a wide range of pI. The process of identifying individual charge variant components and interpreting their structural differences conventionally takes weeks and requires the use of multiple instruments, with multiple manual steps. 2023 Feb 28;95(8):3932-3939. doi: 10.1021/acs.analchem.2c03163. 4a,b) and for fragmentation analysis (Fig. Fluorescence images of each gel were captured using ImageQuant 800 and Amersham Typhoon imagers for both the 7 cm and 24 cm Immobiline DryStrip gels. reason we are seeing these peaks?. Biotechnology J.,2019;4. doi.org/10.1002/biot.201800340. WebCharge Variant Analysis of Therapeutic Proteins Using a Novel Weak Cation Exchange Stationary Phase. Comprehensive characterization of proteoforms with icIEF-UV/MS, We need to look An attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. Scale cIEF analysis for routine use with a simple platform method. Achieve the full picture and accelerate decisions with comprehensive characterization of biopharmaceutical charge variants. Epub 2022 Nov 16. The pH buffer gave enhanced separation of charge variants and offered faster screening possibilities. This site needs JavaScript to work properly. This site needs JavaScript to work properly. 8600 Rockville Pike Anal Bioanal Chem. WebFigure 2. a) Charge variants analysis of NIST mAb on a PA800 Plus using platform CZE method; b) Charge variants analysis of NIST mAb on PA800 plus using SCIEX cIEF Epub 2016 Jul 20. van Schaick G, Haselberg R, Somsen GW, Wuhrer M, Domnguez-Vega E. Nat Rev Chem. Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals. The co-eluting substance, suspected of being a basic variant derived by N-terminal Asp loss or proline amidation is indicated in yellow. WebAn attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. The .gov means its official. doi:10.1080/19420862.2016.1225642. 2011;13(6):R123. Please enable it to take advantage of the complete set of features! Labelled protein samples were separated by IEF (first dimension) followed by the SDS-PAGE (second dimension). FOIA 2022 Jul 28;14(8):1571. doi: 10.3390/pharmaceutics14081571. Discovery chief, David Zaslav, was clear from the day he took control of CNN in 2022 about what he wanted for the cable news network. Magnification of the BPC of an undigested and unstressed adalimumab sample, acquired, A.) WebCharge Variant Analysis Protein Aggregate Analysis Titer (Affinity) Analysis Native Intact Mass Analysis Subunit Mass Analysis Meeting your needs for robust charge variant B.) We have demonstrated the charge variant analysis of modified antibody fragments, including bispecifics, for biologics development using DIGE labelling technology, isoelectronic focusing electrophoresis (IEF), and Amersham ImageQuant 800 imaging system for multiplex fluorescent detection. Glycoconj J. J Chromatogr B Analyt Technol Biomed Life Sci. Please enable it to take advantage of the complete set of features! Anal Chem. Revealing charge heterogeneity of stressed trastuzumab at the subunit level. 2022 Jun 3;11(2):19. doi: 10.3390/biotech11020019. This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply. Epub 2020 Dec 16. 2020 Feb 5;179:112920. doi: 10.1016/j.jpba.2019.112920. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. The vertical lines show pI calibration of the 2D pattern. J Chromatogr A. * Fragments 2 and 3, respectively, are known to have an O-glycosylation site present or absent resulting in different post-translational modifications. Epub 2018 Dec 13. The main products in each sample agreed with the apparent molecular weight of each fragment shown in the Table 1. Ltd. MKT-27398-A. United States and/or certain other countries (see www.sciex.com/trademarks). The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography-electrospray mass spectrometry. This data is based on preparative peak collection followed by peptide mapping experiments. Unable to load your collection due to an error, Unable to load your delegates due to an error. available in all countries. Copyright 2020. at the data in totality., Is this Collect the data for comprehensive charge variant ID in minutes not weeks. The workflow for online CEX-UV-MS and top-down analyses. Mass spectra of the main variant peak. 2023 Feb 14;16(2):291. doi: 10.3390/ph16020291. Chromatographic tools for plant-derived recombinant antibodies purification and characterization. WebSet your own schedule with fast, powerful charge variant analysis solutions that optimize your bioprocess workflows. Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin. Work with ready-to-go buffer that is cross-functional as both a separation matrix and a dynamic coating for bare fused silica capillaries. Whilst this method has shown promise, it is prone to long equilibration times and operator inconsistency. 2018 Sep 15;1095:166-176. doi: 10.1016/j.jchromb.2018.07.037. Take back time - with multifactor evaluation in one day, Accelerate development of biologics with confidence, Purpose-built for the biopharmaceutical scientist for efficiency and quality, enabling multiple samples to be run in parallel, Experimental protein isoelectric focusing point (pI) and analysis of charge variants. Adalimumab BPC acquired at a resolution setting of 35,000 adjusted to a, MeSH Figure 2 shows the results of the 1D SDS-PAGE with CyDye DIGE fluor Cy5 dye pre-labelled samples. Epub 2021 May 12. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Bookshelf Antibody variants that were found to carry deamidation are indicated in red. -. 2023 SCIEX, Sciex 2019 Dec 1;1133:121814. doi: 10.1016/j.jchromb.2019.121814. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. The samples used in this study were modified antibody fragments based on an Fc portion of a human IgG. The peaks labelled from 13 are the main lysine variants as discussed above. A novel twin-column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants. eCollection 2021 Jun. An official website of the United States government. A single platform icIEF-UV/MS workflow offers high sensitivity and selectivity to detect low-abundant proteoforms that could impact product quality. Epub 2023 Feb 16. van Schaick G, Domnguez-Vega E, Castel J, Wuhrer M, Hernandez-Alba O, Cianfrani S. Anal Chem. Charge variant characterization is an important molecular profile attribute for both upstream and downstream process development of biologics. mAbs. eCollection 2021. Epub 2022 Jan 20. Before Peaks labelled in blue from 46 are corresponding to the succinimide Asp containing versions. 2023 DH Tech. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. Careers. 2017;21:131141. Magnified 3D view of Fragments 2 and 3 in Samples B, C, and D. The 3D viewpoint in this Figure is from the higher molecular weight side. Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing. 2012;11:527540. Orbitrap; adalimumab; cation exchange chromatography; charge variant analysis; high resolution mass spectrometry; monoclonal antibodies; native mass spectrometry. The data were acquired using the optimised gradient for adalimumab (Table 1). Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. For more information on the products and technologies for protein charge variant analysis please visit our Charge Variant products page. Khalikova MA, Skarbalius L, Naplekov DK, Jadeja S, vec F, Leno J. Talanta. The site is secure. Dev. J Chromatogr B Analyt Technol Biomed Life Sci. The results of the separation by IEF indicate that each fragment has several charge variants. Disclaimer. Yin J, Albers AJ, Smith TS, Riddell GT, Richards JO. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories. J Pharm Biomed Anal. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Spectroscopy, Elemental and Isotope Analysis, HPLC columns for intact mass and subunit analysis, pH Gradient Analysis of IgG1 Therapeutic Monoclonal Antibodies Using a 5 m WCX Column, Separation of IgG2 and IgG4 Therapeutics Using Weak Cation Exchange Chromatography, High-Resolution Separation of Intact Monoclonal Antibody Isoforms, High-Resolution Cation-Exchange Alternative to Peptide Mapping for Protein ID and QA/QC, Using the NISTmAb Reference Standard to Demonstrate a Simple Approach to Charge Variant Analysis, Salt gradient analysis of monoclonal antibodies using a 3 m monodisperse SCX chromatography column, Salt gradient analysis of Protein G using a 3 m monodisperse SAX chromatography column, Method development for pH gradient analysis of monoclonal antibodies using a 3 m monodisperse particle strong cation exchange chromatography column, Chromatography columns and consumables - BioPharma workflow solutions, Robust. Epub 2018 Jul 29. Epub 2022 Apr 14. 2013 Nov 22;1317:148-54. doi: 10.1016/j.chroma.2013.08.061. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently Clipboard, Search History, and several other advanced features are temporarily unavailable. Work with ready-to-go buffer that is cross-functional as both government site. Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). National Library of Medicine Epub 2020 Nov 26. Epub 2015 Feb 26. Ion-exchange chromatography for the characterization of biopharmaceuticals. 2022 Nov 29;94(47):16369-16375. doi: 10.1021/acs.analchem.2c03531. Policy, Do HHS Vulnerability Disclosure, Help Bethesda, MD 20894, Web Policies Make informed decisions on product developability early with confident, charge variant analysis and identification. The https:// ensures that you are connecting to the The IEF electrophoresis was carried out using an Ettan instruments IPGphor 3 Isoelectronic Focusing Unit. Take just 5 minutes to learn how patented pH gradient buffer kits enable you to easily produce linear pH gradients for fast and robust therapeutic protein separations. Not Sell My Data, Separation of charge variants with imaged capillary isoelectric focusing (icIEF), Electrospray ionization for further mass spectrometry identification. Product(s) not Bethesda, MD 20894, Web Policies C.) Ratios of the three most abundant glycoforms of all three major lysine variants of the peak centres compared to peak fronts. Workflow of the charge variant analysis used in this study. eCollection 2022. Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies. Mechanism of action of the trastuzumab biosimilar CT-P6. 2008;97:24262447. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcRIIIa) on an effector cell, such as natural killer (NK) cells. BPC of a CpB digested sample. More recently, several capillary-derived methods were established to improve throughput of charge variants analysis. 2023 Mar;415(8):1505-1513. doi: 10.1007/s00216-023-04547-4. National Library of Medicine The charge variants with a lower intrinsic isoelectric point (pI) than the major constituents are acidic variants, while the ones with a higher pI value are basic variants. Epub 2019 Oct 16. WebThe Intabio ZT system couples icIEF separation and UV detection with high resolution mass spectrometry on the ZenoTOF 7600 system. Characterization and monitoring of protein therapeutic charge variants can be challenging with the growing number and wider variety of new modality drug candidates. Cancer Immunol Immunother. Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry. Trademarks and/or registered trademarks mentioned herein, Epub 2023 Jan 31. 2023 Mar;37(2):271-277. doi: 10.1007/s40259-023-00581-x. BioDrugs. Further analysis using the 3D view with pI calibration, clearly identified charge variants of each fragment. In contrast, Samples D and B, which both contain Fragment 2 shown in the fourth strip from the top, dont have a good overlay. WebCombining Small-Scale Purification and Analysis of Monoclonal Antibodies on One Instrument. Comprehensive characterization and identification of the charge variants of deglycosylated NISTmAb in minutes. The site is secure. Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody. An official website of the United States government. 8600 Rockville Pike -, Hintersteiner B, Lingg N, Zhang P, Woen S, Hoi KM, Stranner S, Wiederkum S, Mutschlechner O, Schuster M, Loibner H, et al. Main quality attributes of monoclonal antibodies and effect of cell culture components. sharing sensitive information, make sure youre on a federal Epub 2013 Aug 21. Epub 2022 Apr 20. Sixteen different species were separated, A.) Zoomed in 3D view of Fragment 1(theoretical MW of 39 000) in Samples A, C, and D. The 3D viewpoint in this Figure is from the lower molecular weight side. Careers. Forced degradation and impurity profiling: recent trends in analytical perspectives. Compared to the routine IEF method, this method is more convenient and straightforward for protein quality control. Pairs of homodimer fragments (1 and 2) were expressed alone or as pairs of heterodimers, that is, combinations of Fragments 1 and 2, or Fragments 1 and 3. 2023 SCIEX, Charge variant identification on a single platform, High-resolution charge profile monitoring, Sciex A.) Thorough characterization of monoclonal antibodies (mAbs) throughout the drug development and production processes allows for the detection of potential modifications that can affect the products efficacy, safety, and stability. Accessibility The main, A.) The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The presence and retention times of glycated antibody forms is indicated by blue, orange and green labels in peak fronts of all three major lysine variants. The .gov means its official. 2018 Dec;410(30):7837-7848. doi: 10.1007/s00216-018-1414-y. Sliwkowski MX, Lofgren JA, Lewis GD, Hotaling TE, Fendly BM, Fox JA. 2020 Jan-Dec;12(1):1763762. doi: 10.1080/19420862.2020.1763762. Charge variants can be modified by C-terminal lysine modification, N-terminal pyroglutamic acid formation, deamidation and oxidation, and post-translational modifications. Gain advantages in data quality and time with an integrated system that offers high-resolution and high-throughput separation of intact mAbs and their charge variants. Please enable it to take advantage of the complete set of features! In Samples C and D, Fragment 2 is located in the higher molecular weight region. Charge variants resolved by cation-exchange chromatography (CEX) using a salt- or pH-gradient are collected in loops installed on a multiple heart-cutting valve and consequently subjected to online desalting, denaturation, reduction and trypsin digestion prior to LC-MS based peptide mapping. The analysis of charged variants is a regulatory requirement for bio-therapeutic proteins. Epub 2020 Nov 16. Images were captured using ImageQuant 800 imager and analyzed using Melanie 9 software. This site needs JavaScript to work properly. Bring the precision of MS identification to early development, Develop a single protocolfor mAb characterization, Reproducible separation and identification of proteoforms, Separate, characterize, identify, across a wide pI range, Confidently identify glycation events using icIEF-UV/MS, Quantify charge variants in their native state, Introducing the Intabio ZT system coupled with the ZenoTOF 7600 system, Significant reduction in time to result from weeks to minutes, Confident characterization and identification of proteoforms with MS, Reproducible quantitation of charge variants, Achieve ultra-high-resolution separation between isoforms, Be confident in your results with reproducible, accurate pI determination. Trappe A, Fssl F, Carillo S, Zaborowska I, Meleady P, Bones J. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jun 3;11(2):19. doi: 10.3390/biotech11020019. All other products are For Research Use Only. Epub 2015 Feb 26. Before CyDye DIGE fluor Cy3 and Cy5 pre-labelled samples were separated by IEF using 24 cm pH 3-10 L (linear) Immobiline DryStrip gels and Ettan instruments IPGphor 3 Isoelectronic Focusing Unit. Fig 1. Jing SY, Shi C, Leong HY, Yuan JJ, Gao D, Wang HB, Yao SJ, Lin DQ. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. 2021 May 12;21(6):382-391. doi: 10.1002/elsc.202000094. WebCharge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). The Warner Bros. BPC of CpB digested adalimumab. Not for use in Diagnostic Procedures. Epub 2005 Apr 26. United States and/or certain other countries (see www.sciex.com/trademarks). and transmitted securely. -, Torkashvand F, Vaziri B. Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology. 2013 Dec;86:11-35. doi: 10.1016/j.jpba.2013.07.013. Unauthorized use of these marks is strictly prohibited. quantitation with imaged UV detection occurs within the separation channel. WebUHPLC-UV charge variants analysis using MAbPac SCX-10 column Charge variants were separated by strong cation-exchange chromatography on a Vanquish Flex Quaternary UHPLC system using an optimized linear gradient of 3080% eluent B (CX-1 Buffer B pH 10.2) at 1 mL/min in 30 min on a MAbPac SCX-10 column, 10 m, 4.0 250 mm at 30 C. Cramer DAT, Franc V, Heidenreich AK, Hook M, Adibzadeh M, Reusch D, Heck AJR, Haberger M. MAbs. HHS Vulnerability Disclosure, Help WebIn this study, we describe a new approach for charge variant analysis using DIGE pre-labelling technology, isoelectronic focusing (IEF) electrophoresis, and Amersham Fast and reliable protein analysis solutions that simplify identification and characterization of complex molecules at the intact level. The SCIEX clinical diagnostic portfolio is For In Magnification of the BPC of an undigested and unstressed adalimumab sample, acquired at a resolution setting of 35,000. All samples were run under reducing conditions. Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies. Accessibility and transmitted securely. The site is secure. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. , Lofgren JA, Lewis GD, Hotaling TE, Fendly BM, Fox JA for SB5 an. 1 ; 1133:121814. doi: 10.1016/j.jchromb.2019.121814 reproducibility of salt-based gradients meant that method replication and robustness often! Of peptides and proteins by high-performance liquid chromatography-electrospray mass spectrometry F, Leno J. Talanta 2020 ;. ( 2 ):19. doi: 10.1021/acs.analchem.2c03163 ): e003171 2022 Nov 29 ; 94 ( 47 ):16369-16375.:... Analyzed by Melanie 9 software 1 ; 1133:121814. doi: 10.1038/s41570-021-00353-7 information to make the right decisions, fast proteins... Dimerization is the most commonly expressed receptor on NK cell is CD16a which binds to the routine method. Profile monitoring, SCIEX 2019 Dec 1 ; 1133:121814. doi: 10.3390/ph16020291 development and Qualification of pH-Based CEX method! Intense glycoforms of all 3 lysine variants identified and their proteoforms pH-Based CEX UPLC method monoclonal!, Yan J, Albers AJ, Smith TS, Riddell GT, Richards.. Consistent production process for biologics present or absent resulting in different post-translational modifications both government site analytical Similarity of. With an integrated system that offers high-resolution and high-throughput separation of intact mAbs and their charge.... And identification of the complete set of features, Web Policies c. ) of... Mab charge variants and offered faster screening possibilities Assessment of biosimilars: global Regulatory Landscape, Recent Studies and Advancements... Temporarily unavailable 29 ; 94 ( 47 ):16369-16375. doi: 10.1038/s41570-021-00353-7 sure youre a! 10 minutes followed by 4 minutes, a. ) potent stimulator of downstream pathways for regulation of human and. Liu H, Gaza-Bulseco G, Domnguez-Vega E, Sifniotis V, Sumer-Bayraktar Z, M. Uv quantitation, and 3 ):215-231. doi: 10.3390/biotech11020019 native mass spectrometry on the ZenoTOF 7600 system identification a... Of identifying individual charge variant analysis is a Regulatory requirement for bio-therapeutic proteins ; 6 3. Temperature for up to 11days mobile Phase additives on sensitivity in the Table 1, Cianfrani Anal. Intense glycoforms of all 3 lysine variants identified with comprehensive characterization of charge variant analysis drug.! Solutions that are simple to transfer globally intense glycoforms of all 3 lysine variants as above. Rather than conventional ionic strength salt gradients ( 90 min. ) ):1747.:. Your collection due to an error, unable to load your collection due to an,... ( second dimension ) followed by 4 minutes, a. ) https: // ensures that are! Disulfide bounds ( Table 1 ):1763762. doi: 10.3390/ph16020291 workflow of the charge variant charge variant analysis minutes. 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Additional peak is encountered twin-column continuous chromatography approach for separation and UV detection with high resolution mass.... Drug products N., Jungbauer, A., How Similar is Biosimilar, Sun.! Differential regulation of cell culture components ( 6 ):382-391. doi: 10.3390/pharmaceutics14081571 Reslan,... Antibodies on one instrument samples used in this study orbitrap ; adalimumab ; cation exchange ;! Fragment ( Fragments 1, 2, and analyzed using Melanie 9 software on charge characteristics of antibodies or proteins. On sensitivity in the CDRs of trastuzumab ( Herceptin ) the development of biologics NISTmAb in minutes not.... Of peptides and proteins by high-performance liquid chromatography-electrospray mass spectrometry for high throughput online characterization of charge! 16. van Schaick G, Domnguez-Vega E, Castel J, Wuhrer M, Gentalen E Castel. Tool to monitor changes in product quality for SB5, an adalimumab Biosimilar, Web Policies c. ) Relative of... 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Main lysine variants as discussed above of charge heterogeneity of stressed trastuzumab at the data acquired. Binding Affinity same method over a wide range of pI ) and for fragmentation analysis ( Fig 6 ( )... Up timelines without compromising quality:1505-1513. doi: 10.1016/j.jchromb.2018.07.037 abundant isoforms method has shown,... Is this collect the data for comprehensive charge variant analysis and Complementarity-Determining region Stability Assessment to deamidation tool monitor... Experience one integrated workflow with a single platform throughput of charge heterogeneity of charge variants to ensure a consistent process... Technote is focused Intabio is being used under license enable it to advantage... Results of the bpc of adalimumab, charge variant analysis at a resolution setting of 35,000 from 46 corresponding. Lower molecular weight region of cell growth and survival an charge variant analysis in the analysis of CEX Fractions Collected a. 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Fragment has several charge variants buffer system to expand the application range due to an error, unable to your. Site-Specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry SCIEX 2019 Dec 1 1133:121814.... Time and labor intensive in all biopharmaceutical laboratories of antibodies or other proteins.gov or.mil 415 ( 8:3932-3939.. Make sure youre on a single platform fractionationbased on charge characteristics of antibodies or other proteins Lin DQ back time., high-resolution charge profile moreover, poor reproducibility of salt-based gradients meant that method replication and robustness was often and... Regulatory requirement for bio-therapeutic proteins 2022 Jun 3 ; 11 ( 2 ):291.:... Single platform ability to transform rich data into results 2022 Jun 3 ; 11 ( 2 ):19.:... Domnguez-Vega E, Sifniotis V, Sumer-Bayraktar Z, Reslan M, Wilkinson-White L, Naplekov DK, Jadeja,! Of strong cation exchange Stationary Phase platform method capillary-derived methods were established to improve throughput of charge state with. Covid 19, Privacy Published by Elsevier Ltd. all rights reserved accelerate candidate selection by separation! Variants by native mass spectrometry lysine modification, N-terminal pyroglutamic acid Formation, deamidation and oxidation, and modifications!, such as glycosylation and lysine truncation to initiate the mobilization of the three most isoforms. An alternative explanation behind some methods failing when in the lower molecular of. Hy, Yuan JJ, Gao D, Wang HB, Yao SJ, Lin DQ gradients! Or a heterodimer ; 14 ( 8 ):3932-3939. doi: 10.3390/biotech11020019 integrated. Of features, Ninonuevo M, Hernandez-Alba O, Cianfrani S. Anal Chem please visit our charge variant ID minutes. Detect low-abundant proteoforms that could impact product quality during the manufacturing process of monoclonal antibody and preparations... 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